Atomic force microscopy analysis of progenitor corneal epithelial cells fractionated by a rapid centrifugation isolation technique.

PURPOSE

To investigate the use of atomic force microscopy (AFM) to image the three groups of corneal epithelial cells fractionated by a novel rapid centrifugation isolation technique.

METHODS

Epithelial cells harvested from primary cultures of rabbit limbal rings were centrifuged onto uncoated dishes, first at 1400 rpm and then at 1800 rpm. The adherent cells after centrifugation at 1400 rpm (ATC1), the adherent cells at 1800 rpm (ATC2) and the non-adherent cells at 1800 rpm (NAC) were investigated for BrdU retention and were subjected to contact mode AFM and Transmission Electron Microscopy (TEM).

RESULTS

Compared with unfractionated cells, the ATC1 group, accounting for about 10% of the whole population, was enriched in BrdU label-retaining cells. There were dramatic overall shape, surface membrane and intra-cellular ultrastructure differences noted among ATC1, ATC2 and NAC populations. The whole cell roughness measurements were 21.1±1.5 nm, 79.5±3.4 nm and 103±4.6 nm for the ATC1, ATC2 and NAC groups, respectively. The mero-nucleus roughness measurements were 34.2±1.7 nm, 13.0±0.8 nm and 8.5±0.5 nm in the ATC1, ATC2 and NAC populations, respectively.

CONCLUSIONS

AFM was found to be a good tool for distinguishing among the three groups of cells. BrdU label retention, the AFM parameters and TEM together suggest that the ATC1, ATC2 and NAC populations may be progenitor corneal epithelial cells, transit amplifying cells and terminal differentiation cells, respectively.